Phylogenomic analysis of Syngnathidae reveals novel relationships, origins of endemic diversity and variable diversification rates

Background Seahorses, seadragons, pygmy pipehorses, and pipefishes (Syngnathidae, Syngnathiformes) are among the most recognizable groups of fishes because of their derived morphology, unusual life history, and worldwide distribution. Despite previous phylogenetic studies and recent new species descriptions of syngnathids, the evolutionary relationships among several major groups within this family remain unresolved. Results Here, we provide a reconstruction of syngnathid phylogeny based on genome-wide sampling of 1314 ultraconserved elements (UCEs) and expanded taxon sampling to assess the current taxonomy and as a basis for macroevolutionary insights. We sequenced a total of 244 new specimens across 117 species and combined with published UCE data for a total of 183 species of Syngnathidae, about 62% of the described species diversity, to compile the most data-rich phylogeny to date. We estimated divergence times using 14 syngnathiform fossils, including nine fossils with newly proposed phylogenetic affinities, to better characterize current and historical biogeographical patterns, and to reconstruct diversification through time. We present a phylogenetic hypothesis that is well-supported and provides several notable insights into syngnathid evolution. We found nine non-monophyletic genera, evidence for seven cryptic species, five potentially invalid synonyms, and identified a novel sister group to the seahorses, the Indo-Pacific pipefishes Halicampus macrorhynchus and H. punctatus. In addition, the morphologically distinct southwest Pacific seahorse Hippocampus jugumus was recovered as the sister to all other non-pygmy seahorses. As found in many other groups, a high proportion of syngnathid lineages appear to have originated in the Central Indo-Pacific and subsequently dispersed to adjoining regions. Conversely, we also found an unusually high subsequent return of lineages from southern Australasia to the Central Indo-Pacific. Diversification rates rose abruptly during the Middle Miocene Climate Transition and peaked after the closure of the Tethys Sea. Conclusions Our results reveal a previously underappreciated diversity of syngnathid lineages. The observed biogeographic patterns suggest a significant role of the southern Australasian region as a source and sink of lineages. Shifts in diversification rates imply possible links to declining global temperatures, the separation of the Atlantic and Pacific faunas, and the environmental changes associated with these events. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-022-01271-w.


Calibration Densities
The Hedman approach [107] generates a distribution of probable time of origin for a node based on ages of a sequence of outgroups, given (i) the minimum age of the calibrated node, (ii) a sequence of stratigraphically consistent of older fossil outgroup to that node, and (iii) a hard maximum bound on the ages. More distant outgroups have to appear earlier than more proximate outgroups to the focal node, i.e. the fossil record is stratigraphically consistent with the tree [ We extended this outgroup sequence with stratigraphically consistent calibrations included in our own set of fossil-based minimum ages for Syngnathiformes. We also include one additional fossil, for which the phylogenetic position could be specified as a crown Syngnathidae but which was not used as calibration itself (see Additional node A). The outgroup sequence for each calibration point is given under "Outgroup age sequence". The outgroup sequence requires a maximum hard bound, which was set in the Carboniferous (322.8 Ma, stem neopterygian †Discoserra, [108]), far exceeding any of the divergences investigated here.
In order to estimate the calibration densities for each of the 14 calibration nodes, we applied the outgroup sequence with an R script from [108]. The obtained mean and 95% confidence interval was applied on the prior distribution of the node age in BEAST2 (specific settings given under "Settings in BEAST" for each node).

Fossil Justifications
Additional Node A: Crown Syngnathidae Because a central goal of the fossil calibrations was to infer the ages of nodes within Syngnathidae, we did not place an age constraint on the MRCA of Syngnathidae. Instead, we use the information of this crown Syngnathidae fossil as part of the outgroup sequence to define the prior distribution of node ages for syngnathid calibrations (Nodes [8][9][10][11][12][13][14]. Fossil taxon: †Prosolenostomus lessinii Blot 1980 Material: description of holotype, IG 37597 Vérone. Phylogenetic placement: MRCA of Syngnathidae Placement justification: The original description placed the fossil as a stem Solenostomidae [12]. The presence of a number of Syngnathidae features, which are missing in Solenostomidae, is evidence for a placement as a crown Syngnathidae: presence of dermal bony rings (stellate plates in Solenostomidae); single dorsal fin (double in Solenostomidae); pelvic fins absent (present in Solenostomidae) [13,46,70]. Because of the absence of apomorphies for Syngnathidae relative to the fossil, it cannot be placed on the stem of Syngnathidae (which would also make it synonymous with Node 7). The presence and location of the brood pouch is unknown, which makes it hard to know if the fossil could be placed on the stem of Nerophinae or Syngnathinae or within one of these groups. We therefore exclude the fossil as a node calibration point but include its information content with respect to middle Eocene occurrences of Syngnathidae in the outgroup sequence for syngnathid calibrations. Stratigraphy: middle Eocene, Ypresian, Pesciara or Monte Bolca, Italy Minimum age: 48.5 Ma Age justification: The deposits from Monte Bolca's two principal Lagerstätten correspond to large parts of the Shallow Benthic Zone (SBZ) 11 of the late Ypresian Stage and have been dated to 50.7-48.5 Ma [14]. Discussion: Equivalent fossils from the same deposit may be "Syngnathus" heckeli de Zigno 1874a and "Syngnathus" bolcensis de Zigno 1874b. Their attribution to the extant genus Syngnathus should be verified, as Syngnathus functioned as a catchall for any pipefish in the early paleontological literature.

Age justification:
The stratigraphic layer bridges the late Paleocene (NP9) into the early Eocene (NP10) and has been dated to 55.964-55.728 Ma [22]. Note: The lower Eocene †Protoramphosus parvulus Danil'chenko 1968 is an equivalent fossil calibrating the same node based on the present sampling and is slightly younger (lower Eocene, Ypresian, Danatinsk Formation, Turkmenistan, 54.17 Ma [23]). The fossil can be placed on the stem of MNC based on elongate anterior vertebrae, the large dorsal spine, and the structure of the head [13]. To our knowledge, this is the first time this fossil was used in calibrating a time tree (see Placement justification  [26]. †Solenorhynchidae share five characteristic features with extant Solenostomidae: brood pouch formed by pelvic fins; body covered with stellate plates; pseudospines on dorsal and pelvic fins; pelvic fins have one pseudospine and six rays; no plostcleithrum [13,26]. They are distinguished from crown Solenostomidae by pelvic fins that insert anterior of the spinous dorsal fin [26], placing them on the stem of Solenostomidae. Stratigraphy: middle Eocene, Ypresian, Pesciara or Monte Bolca, Italy Minimum age: 48.5 Ma Age justification: The deposits from Monte Bolca's two principal Lagerstätten correspond to large parts of the Shallow Benthic Zone (SBZ) 11 of the late Ypresian Stage and have been dated to 50.7-48.5 Ma [14]. Outgroup  , placing it in Nerophinae. It has spiny ridges with denticulate margins on the body rings, which is characteristic of members of the clade Doryrhamphini+Microphini+Heraldia+Maroubra (Table S2). Their sister group Nerophini have more smooth bodies without denticulate margins on their body rings. The fossil also has a longitudinal opercular ridge [27], which is also present in members of the spiny ridge clade except for some species of Microphis [43], while Nerophini lack opercular ridges in adults, albeit present in juveniles of Leptoichthys [43]. The original description of the fossil argued for an affiliation with Maroubra [27] but the remaining listed characters are not apomorphic for Maroubra or for others within the spiny ridge group (Table S2): moderate elevation of dorsal medial snout ridge without spines or denticles (also present in Microphini), a caudal fin with 10 rays (also present in Doryramphini and in Choeroichthys in Microphini), pouch plates covering the brood pouch (not present in Maroubra). We therefore argue for a stem group placement on the spiny ridge clade based on the apparently apomorphic spiny body ridges and opercular ridges. Stratigraphy: lower Oligocene, Rupelian, Pshekhskiy horizon of the lower Maikop series, Adygeya,  Danil'chenko 1960 [70, 29]) from the same stratum and locality may calibrate the same node as †Maroubrichthys serratus but it is less completely preserved. We discuss some points that may help in a future placement. The fossil has a fully developed anal fin [70] unlike all extant syngnathids that have minute anal fins. The position of the brooding area is unknown [70], which leaves the possibility that the species belongs to Syngnathinae. On the other hand, the fossil has spines on rings and a longitudinal opercular ridge like members of the Nerophinae spiny ridge clade and †Maroubrichthys serratus. To our knowledge, this is the first time this fossil was used in calibrating a time tree. It can be further assigned to the clade of Doryramphini+Microphini+Heraldia+Maroubra due to its spines distally on the body rings and the presence of a longitudinal opercular ridge (Table S2). Additional characters that are preserved are not apomorphic for more inclusive clades: the prominent caudal fin is found in Doryrhamphini, Microphini and Heraldia but absent in Maroubra; the number of rings is overlapping with most groups (40-44 body rings, 15-17 trunk rings, [30], (Table S2). The fossil has an elongated pectoral ring, which is characteristic of Doryrhamphini, and keeled scutella, which are characteristic of Microphini. Given this occurrence of both keeled scutella and the hypertrophied pectoral ring in the same species, these traits may not be apomorphic of the crown groups but of Microphini+Doryrhamphini, with subsequent loss in one of these groups. We therefore place the fossil on the stem of Microphini+Doryrhamphini. . This interpretation may have been guided by a previous phylogeny that showed pygmy pipehorses as sister to seahorses [52]. Taxonomically denser phylogenies showed a more complicated relationship with several pipefish lineages that are more closely related to the seahorses and "pygmy pipehorses" than they are to each other ([44]; this study). These new findings require a reinvestigation of the affiliation of †Hippotropiscis frenki. The thorough morphological description by [47] allows for a placement on the stem pygmy pipehorses (Idiotropiscis including Acentronura tentaculata) rather than with seahorses. Like pygmy pipehorses the fossil has pouch plates (absent in seahorses) and an obtuse angle of the head to the body (head in a sharp angle to the trunk in seahorses). Within pygmy pipehorses, the fossil shares the discontinuous tail and trunk ridges with members of Idiotropiscis but not with Acentronura. Given the inclusion of Acentronura within Idiotropiscis, the discontinuous tail and trunk ridges could be a stem trait of pygmy pipehorses, with a loss in Acentronura. The elevated frontal ridge of the fossil and Idiotropiscis lumnitzeri and I. australe, but not Acentronura or Cylix, suggests at least two gains given the present phylogeny and is therefore not useful in placing the fossil.  [7][8][9][10][11][12][13][14] and more consistent with the range of the other non-pygmy seahorses (range 11-33). As their names implies, pygmy seahorses also have a diminutive body size and the fossils are tiny as well (6-15 mm). However, body size reduction also happened within the nonpygmy clade of seahorses (e.g. H. zosterae). The interpretation of †Hippocampus slovenicus as a crown pygmy seahorse is therefore uncertain. A stem position seems more appropriate given the shared similarities with non-pygmy seahorses, which effectively places the fossil on the crown of all Hippocampus in node dating. Stratigraphy: middle Miocene, lower Sarmatian, Coprolithic horizon, Tunjice Hills, Slovenia Minimum age: 11.6 Ma Age justification: The Sarmatian stage in the area covered a time span between 11.6-12. , the dorsal fin supported by 2 trunk and 1 tail ring (shared with at least 25 species), and the narrow head with low coronet in line with the arc of the neck (low coronets are shared by 15 species [51]). While the fossil clearly appears to be a member of the crown of non-pygmy seahorses due to these shared characteristics, none of the characters are diagnostic to place it among an extant non-pygmy seahorse clade.
The fossil shares 11 trunk rings with most non-pygmy seahorses (range 11-12), distinct from the sister group H. jugumus (13 trunk rings). We therefore place the fossil on the crown of non-pygmy seahorses excluding H. jugumus.
The fossil was previously used on the crown group of Hippocampus, defined by the sampling as the MRCA of H. abdominalis and other seahorses [11]. Therefore, the placement of the fossil was the same as here, although we argue that it is not a crown member of Hippocampus in its entirety. Here, by including pygmy seahorses and H. jugumus, we show that the crown group of Hippocampus is larger.

Fossil taxon only used in biogeographic analysis
In addition to the 14 fossils used for fossil calibration, we used †Protoramphosus parvulus (Node 5b in Fig. 5a), which was not used for node calibration because it calibrated the same node as another fossil (Node 5, MRCA of Centriscidae), but could inform the biogeographic reconstruction applied to the lineage leading to Macroramphosus.   Endemic to southwestern Australia.

Taxonomy
Our sampling identified (1) potential new species, (2) species in previous synonymy, (3) nonmonophyletic genera (including implications for synonymous genus names), (4) extended biogeographic realms. We list the current evidence below and recommend taxonomic actions if sufficient evidence exists. Marquesas Islands. Given the short branch lengths separating the two specimens, the putative species is likely distributed at least across the Central Indopacific areas that lie between the two sampling localities. Comparison to publicly available COI barcodes also shows two other specimens from the Seychelles (99.4% identity, SAIAB 78058-T455, SAIAB 78058-T456) from the same lot as our Seychelles specimen (SAIAB 78058), while other Doryrhamphus species are at least 8% divergent. A2) Doryrhamphus excisus sp2. This putative species has been sampled from the Philippines.
Comparison to publicly available COI barcodes shows this species is also present in Lizard Island, Australia (99.4% identity, LIFSA041-08), with the caveat that the obtained COI fragment from our specimen is short (292 bp). A3) Doryrhamphus excisus sp3. This putative species is represented by a specimen from Guam and the Northern Mariana Islands. The two specimens have a COI pairwise distance in available COI barcodes of at least 6.5% to other Doryrhamphus species. B) Halicampus dunckeri is sampled with two specimens from the Philippines here, which are sister groups but are separated by long branches. Without material from the type locality in Indonesia, it cannot be discerned which lineage represents the nominal H. dunckeri. C) Hippichthys penicillus is represented by two specimens from Kuwait separated by long branches, while one of the lineages is also represented by a specimen from Australia. Without material from the type locality in Malaysia, it cannot be discerned which lineage represents the nominal H. penicillus.

D)
Hippocampus mohnikei sp2 was suggested as potentially cryptic based on barcoding data [51], and we also find a substantial divergence in nuclear DNA. Publicly available COI barcodes show that our specimen from Malaysia is >99% identical in sequence to specimens from India (GenBank accession numbers MN595217, MK330041, MN595216). The occurrence of H. mohnikei in Indian waters was interpreted as a range expansion of H. mohnikei [62] but with a minimum divergence in COI of 7.8% from nominal H. mohnikei, the interpretation as a separate species may be more justified. E) Stigmatopora nigra sp2. Stigmatopora was shown to have strong genetic structure across its range, possibly warranting description of 2 new species, in addition to the nominal S. nigra [61]. We sampled one of these undescribed species from New Zealand (S. nigra sp2 in the main figures which corresponds to "Stigmatopora_nigra2" in [61]) and confirmed some genetic distance to the nominal S. nigra (here sampled from Queensland, Australia).   is the type species by original designation and was described from Port Jackson, New South Wales. Our specimen of F. cinctus is from the close-by Nelson Bay, New South Wales. We therefore suggest that

(4) Extensions of known ranges or confirmed wide distributions
Cases where our sampling extended known biogeographic ranges or confirmed wide ranges, in alphabetic order.
Choeroichthys suillus has only been recorded from northern Australia from Perth across north Australia to southern Queensland [43] but the phylogeny here shows a specimen from Palau being sister to a specimen from Queensland.
Corythoichthys polynotatus inhabits coastal waters in the Philippines, Indonesia, and Palau [43] but our phylogeny shows the species extends also to Guam.
Cosmocampus banneri has an usually wide range across much of the Pacific [43], which we confirm with samples from Japan close to the type locality, from the Red Sea and the Philippines. We include specimens from the Philippines but also Japan, which extends the known range north.      Fig. S12. Calibrated phylogeny from BEAST2 with the fossil calibration set excluding the constraint on the nodes within seahorses (Hippocampus). The arrows indicate the shift of node estimates compared to the full fossil calibration set, if the age difference of a node is more than 1 Ma. Across all nodes, the nodes are on average very slightly younger (mean=-0.63, median=-0.47). The maximum difference between nodes of the two trees is on the most recent common ancestor of seahorses, which is -2.3 Ma younger in this analysis.    Tables   Table S1. Number of described and sampled species per genus for Syngnathidae. The sister group Solenostomidae is also included because this study sampled several species. If a genus was paraphyletic, missing species were assigned based on taxonomic characters. Where the species count deviates from published references, see Notes at the bottom of the table for justifications. Most concern the synonymy of species, which largely follows [40,43,51,116]. Cases in which our data shows deviations are annotated in Notes.  Table S2. Comparison of diagnostic characters between the fossils and extant members of Nerophinae.